Publication Date

2018-08-21

Availability

Embargoed

Embargo Period

2019-08-21

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2018-07-24

First Committee Member

Justin Percival

Second Committee Member

Marjana Tomic-Canic

Third Committee Member

Tongyu Cao Wikramanayake

Fourth Committee Member

Antoni Barrientos

Fifth Committee Member

Tasuku Akiyama

Abstract

Chronic wounds affect more than 6.5 million people each year in the United States and represent a major healthcare burden for patients and healthcare professionals. Chronic wounds are a complex multi-factorial disease with limited treatment options, and new therapy approaches are desperately needed. The long-term goal of this project is to provide a novel therapeutic approach for chronic wounds using modulators of cholesterol synthesis, statins. Re-purposing FDA approved drug(s) is a promising strategy that facilitates rapid clinical translation from bench-to-bedside. Skin synthesizes cholesterol, the precursor to all steroid hormones. Our laboratory has shown that epidermis uses cholesterol to synthesize, cortisol, a glucocorticoid hormone (GC) that inhibits keratinocyte migration and wound healing. Cortisol acts through the glucocorticoid receptor (GR) leading to its phosphorylation and activation, resulting in its nuclear translocation where it regulates expression of target genes including ones involved in wound healing. Also, we have shown that an intermediate in the cholesterol biosynthesis pathway, farnesyl pyrophosphate (FPP), acts as a ligand for the GR and inhibits wound healing. Hence, inhibiting the cholesterol biosynthesis pathway in skin may result in suppression of GR activation through reduction of both of its ligands, cortisol and FPP, thus restoring healing. Statins act by targeting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthesis pathway, resulting in lower cholesterol levels. Statins are a widely used class of drugs with a high safety profile and it is expected that the use of statins will increase in the future. Recent studies have suggested that statins can exert independent pleiotropic effects including anti-inflammatory, antibacterial properties, decreased oxidative stress, improved endothelial function, and improved healing outcomes as well as regulate expression of non-coding RNAs involved in regulation of proliferation and inflammation. All of these processes are deregulated in chronic wounds, which make statins attractive treatment modalities. However, in order to implement such therapy, the mechanisms by which statins exert diverse beneficial effects to promote wound healing need to be better characterized. This thesis, therefore, tested the hypothesis that the mechanisms by which statins promote epidermal wound healing is by inhibiting synthesis of GR ligands, cortisol and FPP. The major questions asked were 1) do statins reverse the effects of GR-mediated wound healing inhibition? 2) do statins inhibit phosphorylation and activation of GR by inhibition of HMG-CoA reductase? and 3) given that statins are able to exert pleiotropic effects, what are possible mechanisms by which statins promote epidermal healing that are GR independent? To answer these questions, we utilized porcine in vivo wound model, in vitro keratinocyte scratch assay, organotypic 3D skin cultures and tissue obtained from patients with DFUs and assessed the mechanisms and functional effects of mevastatin on reversing GR inhibitory effects on keratinocyte migration. In addition, to gain a better understanding of the full therapeutic potential of statins, we performed RNA-seq on mevastatin treated primary human keratinocytes to identify novel mechanisms of statin action in keratinocytes that promote wound healing. Here, we demonstrate that mevastatin reverses GC inhibitory effects and promotes healing by using in vitro wound healing assays, human ex vivo and porcine in vivo wound models, and DFU tissue obtained from patients. Moreover, we measured cortisol levels by ELISA and found mevastatin inhibited cortisol synthesis in keratinocytes and biopsies from patients with diabetic foot ulcers (DFU). Importantly, topical mevastatin stimulated epithelialization in vivo and also reversed FPP-mediated induction of the GR target, c-Myc (a biomarker of nonhealing wounds) in human wound models. Importantly, mevastatin reversed c-Myc overexpression in DFUs. In addition, we identified novel mechanisms of statin action in promoting wound healing. We found that mevastatin induced expression of the long noncoding RNA Gas5 that blocks c-Myc expression, which was confirmed by overexpression studies. Furthermore, next generation sequencing revealed statins inhibited cell proliferation while inducing cell migration, suggesting statins may shift the chronic wound phenotype from a hyperproliferative to a migratory phenotype to restore healing. Interestingly, mevastatin restored EGF signaling, a potent stimulator of keratinocyte migration, through modulation of caveolae in DFU tissue. We conclude that topical mevastatin accelerates wound closure by promoting epithelialization via multiple mechanisms: modulation of GR ligands and as well as through exerting pleiotropic effects that include induction of the long non-coding RNA, Gas5, as well as restoring EGF signaling to promote wound healing. Collectively, this work identified novel mechanisms by which statins reverse GR-mediated effects as well as unique statin-regulated pathways suggesting that topical statins may have considerable therapeutic potential as a treatment for patients with chronic wounds. In addition to wound healing, topical statin-therapy provides a potential novel therapeutic strategy for several dermatological conditions. Understanding the mechanisms by which statins promote epidermal healing and affect keratinocyte function will provide a basis for directing future therapies by identifying additional beneficial effects and improving current therapies aimed not only at wound healing disorders, but other dermatological diseases as well. Cutaneous wound healing represents a paradigm of tissue repair mechanisms. Thus, understanding how statins regulate this process will be applicable to other tissues beyond just skin, such as cornea, intestine, or lung. This work has led to new knowledge and understanding of the statin-mediated mechanisms that regulate wound healing and offer a potential novel treatment for chronic wounds that can be rapidly translated to clinical use.

Keywords

diabetic foot ulcers; wound healing; glucocorticoids; long non-coding RNAs; human epidermal keratinocytes

Available for download on Wednesday, August 21, 2019

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